1. Field of the Invention
The invention relates to a process for the preparation of a plasma product which is virtually free from active viruses or of a concentrate of blood coagulation factors II, VII, IX and X which is virtually free from such viruses, by heating in the presence of stabilizers, and to a product of these factors which is thereby prepared. Such products can be used for the treatment of blood coagulation disorders.
2. Description of the Prior Art
Coagulation of blood is a complex process which proceeds in stages and is triggered of by various physiological and pathological causes, its course depending on about 20 promoting or inhibiting factors. Disorders in blood coagulation, some of which manifest themselves as diseases, occur as a result of a reduction or increase in these blood coagulation factors.
Concentrates of factors II, VII, IX and X are suitable for the treatment of various congenital or acquired disorders in the synthesis of these factors. The treatment of patients with concentrates of these factors has hitherto been associated with the risk of transmission of viruses and, in particular, hepatitis.
Albumin is hepatitis-safe if it is heated to 60xc2x0 C. in aqueous solution and in the presence of stabilizers (Gellis, S. S. et al., J. Clin. Invest. (1948) 27, 239). It is therefore to be assumed that a concentrate of factors II, VII, IX and X heated in the presence of suitable stabilizers is likewise hepatitis-safe.
German Offenlegungsschrift 2,916,711 describes a process for the heat-stabilization of other coagulation factors in aqueous solution by addition of an amino acid and a mono- or oligo-saccharide or sugar-alcohol.
However, inactivation of factors II, VII, IX and X during heating cannot be prevented in this way. The observation that factors II and VII can be protected from thermal inactivation in aqueous solution by chelating agents (German Patent 3,043,857 A1) and factors IX and X can be protected by calcium (German Patent 3,045,153 A1). was therefore an advance. However, neither process allows the preparation of a product of all four factors in one operating process, as is desirable, since all four factors are reduced in cases of vitamin K deficiency or under therapy with oral anti-coagulants and must therefore be replaced at the same time.
Accordingly, there was the object of discovering a process for stabilizing an aqueous solution containing factors II, VII, IX and X from thermal inactivation.
Surprisingly, it has now been found that an aqueous solution containing all four factors II, VII, IX and X can be protected From the adverse consequences of heat treatment by addition of calcium ions and a chelating agent. If appropriate, antithrombin III (AT) and heparin can be added in order to prevent activation of factors II and VII. The preconditions were thus provided for preparing these four factors, with a high purity, and yield arid containing no active viruses, in one operation.
The chelating agent is added in addition to the citrate already present.
It is surprising that a mixture of calcium ions and chelating agent effects the desired protection of all factors II, VII, IX and X from toss in activity by heating, although, according to German Patent 3,043,857, a chelating agent is advantageous for protecting factors II and VII, whitst calcium ions are used for the same purpose for factors IX and X (German Patent 3,045,153), and chelating agents also form complexes with calcium ions. In each case only the two corresponding factors should therefore be protected from degradation by heat, and not all four, depending on whether the chelating agent or the calcium ions are present in excess.
The invention thus relates to a process for the preparation of a virtually virus-free and hepatitis-safe product of blood coagulation factors II, VII, IX and X by heating an aqueous solutions if appropriate in the presence of an amino acid and/or a saccharide or sugar-alcohol, which comprises heating the solution in the presence of calcium ions and a chelating agent and, if appropriate, antithrombin III and/or heparin.
Such a solution can be a solution containing these factors or a concentrate containing these, as well as plasma or a plasma or placenta fraction.
The optimum concentration of calcium ions is in the range from 1 to 50 mmol/l, preferably 25 to 50 mmol/l.
Examples of suitable salts which supply calcium ions are the chloride, acetate or nitrate, and all water-soluble calcium salts of sugar-acids, such as gluconic acid or lactonic acid. The chloride and acetate are preferably used.
Examples of suitable chelating agents are: ethylenediamino-tetraacetic acid (EDTA), ethylene glycol bis-(2-aminoethyl ether)-tetraacetic acid (EGTA), diaminocyclohexane-tetraacetic acid (CDTA), diaminopropane-tetraacetic acid and nitrilotriacetic acid, and, in particular, soluble alkali metal salts thereof.
Aliphatic aza-tri- or -tetra-carboxylic acids with 6-20 carbon atoms and 1 or 2 nitrogen atoms and soluble alkali metal salts thereof and, in particular, the sodium salts of ethylenediaminotetraacetic acid (EDTA) or ethylene glycol bis-(2-amino-ethyl ether)-tetraacetic acid (EGTA) are preferably used. The optimum concentration of the chelating agents is 1 to 20 mmol/l, preferably 5 mmol/l.
Mixtures of 25 mmol/l of calcium with 5 mmol/l of EDTA have proved particularly suitable.
Antithrombin III is employed in a concentration of 0.05-2 units/ml, based on the activity of 1 ml of citrated mixed plasma (1 unit), and together with heparin in concentrations of 0.5-2 ) USP units/ml, preferably 0.2 unit of antithrombin III with 2 USP units of heparin.
In the presence of a mixture of calcium ions and a chelating agent and, if appropriate, the antithrombin III-heparin complex, the aqueous solution of the coagulation factors can be heated until, according to the current state of knowledge, transmission of viruses, and in particular hepatitis pathogens, can be virtually excluded. This particularly applies if the pasteurization is combined with adsorption and precipitation processes, in which the active compound remains in the supernatant liquor and the hepatitis viruses can be separated off together with the insoluble precipitate. A product which has been kept at about 60xc2x0 C. in aqueous solution for at least 10 hours is at present regarded as virtually hepatitis-safe, especially if the starting material is human tissue fluid in which hepatitis viruses have not been detected by a third generation test.
A particularly preferred embodiment of the invention comprises adding to a solution containing all four factors, preferably plasma or a plasma or placenta fraction, 0.2-2 units/ml of antithrombin III, 2-20 USP units/ml of heparin, 25-50 mmol/l of calcium ions and 1-20 mmol/l of EDTA, 1-3 mol/l of at least one of the amino acids glycine, alpha- or beta-alanine, lysine, leucine, valine, asparagine, serine, hydroxyprotine, protine, glutamine or alpha-, beta- or gamma-aminobutyric acid, but preferably glycine, and 20 to 60 g/100 g of solution of a mono- or oligo-saccharide or sugar-alcohol, preferably 1 to 3 mol/l of glycine and 20 to 60 g/100 g of solution of sucrose, heating the mixture to a temperature of 30xc2x0 C. to 100xc2x0 C., preferably 60xc2x0 C. to 100xc2x0 C. and keeping it at this temperature for 1 minute to 48 hours, preferably 8-12 hours, the shortest time being matched with the highest temperature and vice versa.
A pH value of 6 to 8 is maintained. A virtually virus-free product of factors II, VII, IX and X is obtained in this manner.
Depending on the solubility of the calcium salt, the amino acid or the carbohydrate, the corresponding concentrations of 0.3 and 3.0 mol/l and 60 g/100 g can be extended to higher concentrations if the calcium salt, the aminoacid or the carbohydrate have a correspondingly higher solubility at the desired temperature. The temperature treatment can also be carried out in several successive steps.
A hepatitis-safe product is achieved with the preferably used combination of antithrombin III, heparin, calcium chloride and EDTA with glycine and sucrose by heating.
In order to obtain a virus-free plasma which contains factors II, VII, IX and X in natural and active form, a mixture of a chelating agent, preferably EDTA, and calcium ions, preferably in amounts such that these are present in concentrations of 5 mmol/l of EDTA and 25 mmol/l of calcium chloride, is added to the plasma and the mixture is heated to 60xc2x0 C. in a sucrose (60 g/100 g)/glycine (2 mol/l) mixture and kept at this temperature for 10 hours.
In order to obtain a natural and active concentrate of factors II, VII, IX and X, a mixture of a chelating agent, preferably EDTA and calcium ions, preferably in concentrations of 5 mmol/l of EDTA and 25 mmol/l of calcium chloride, is added to the plasma or placenta fraction, advantageously a fraction in which the factors to be stabilized are concentrated. After addition of antithrombin III, preferably 0.2 unit/ml, and heparin, preferably 2 USP units/ml, the mixture is kept in sucrose (60 g/100 g)/glycine (2 mol/l) and heated 10 h to 60xc2x0 C.
Such a fraction is obtained, for example, by the method of Soulier et al., Thrombosis Diath. Haemorrh. Suppl. 35, 61 (1969). For this, plasma which has been obtained from blood anti-coagulated with 0.01 mol/l of EDTA is adsorbed onto calcium phosphate and the solid is centrifuged off. The factors are thereby bonded quantitatively to the adsorbent and can be isolated by eluting several times with 0.2 mol/l of tri-sodium citrate. The combined eluates are further purified by combined alcohol and acetic acid precipitation at temperatures of xe2x88x928xc2x0 C. to +4xc2x0 C. The factors are thereby at the same time concentrated.
The concentrate is taken up in a suitable buffer, advantageously sodium chloride/sodium citrate containing 0.06 and, respectively, 0.02 mol/l and with a pH of 7.5.
The coagulation factors can be recovered from the heated solution and purified by precipitation with ammonium sulfate at 30-45% saturation, adsorption of the supernatant liquor onto 0.4 to 1 g/100 ml of calcium phosphate and elution.
The monitoring of the measures for the enrichment and purification of factors II, VII, IX and X is familiar to the expert from the knowledge of determination methods for the substances in question. Using these monitoring methods, the process conditions can be controlled in respect of a satisfactory yield and satisfactory purity of the product.
Factor II can be determined, for example, by the method of Koller, F. et al., Dtsch. med. Wschr. 81, 516 (1956). For this, one part, for example 0.1 ml, of factor II-deficient plasma and one part of dilute normal plasma are mixed. This mixture is kept at +37xc2x0 C. for 30 seconds. Thereafter, two parts of calcium-containing thromboplastin, for example prepared according to German Patent 2,356,493, are added and the time which elapses before a coagulum appears is determined. For a quantitative determination, the coagulation time resulting from the solution containing factor II is read off with reference to a calibration curve obtained with a normal plasma dilution series.
One unit of factor II corresponds to the factor II activity of 1 ml of normal plasma.
Factor VII can be determined, for example, by the method of Koller, F. et al., Acta haemat. 6, 1 (1951). For this, one part, for example 0.1 ml, of factor VII-deficient plasma and one part of dilute normal plasma are mixed. This mixture is kept at +37xc2x0 C. for 30 seconds. Thereafter, two parts of calcium-containing thromboplastin, for example prepared according to German Patent 2,356,493, are added and the time which elapses before a coagulum appears is determined. For quantitative determination, the coagulation time result in from the solution containing factor VII is read off with reference to a calibration curve obtained with a normal plasma dilution series.
One unit of factor VII corresponds to the factor VII activity of 1 ml of normal plasma.
Factor IX is determined, for example, by the following method:
1 part, for example 0.1 ml, of partial thromboplastin, for example prepared according to German Auslegeschrift 2,316,430, is mixed with one part of factor IX-deficient plasma and one part of dilute normal plasma. This mixture is kept at 37xc2x0 C. for 6 minutes. After addition of one part of a 0.025 molar calcium chloride solution which has been prewarmed to 37xc2x0 C., the time which elapses between the addition of the calcium chloride solution and the appearance of a coagulum is determined. For quantitative determination, the coagulation time resulting from the solution containing factor IX is read off with reference to a calibration curve obtained with a normal plasma dilution series.
1 international unit (=1 IU) of factor IX corresponds to the factor IX activity of 1 ml of normal plasma.
Factor X can be determined, for example, by the method of Duckert et al., Blood Coagulation, Hemorrhage and Thrombosis, Ed. Tocantins, L. M. and Kazal, L. A. (1964). For this, one part, for example 0.1 ml, of factor X-deficient plasma and 1 part of dilute normal plasma are mixed. This mixture is kept at +37xc2x0 C. for 30 seconds. Thereafter, two parts of calcium-containing thromboplastin, for example prepared according to German Patent 2,356,493, are added and the time which elapses before a coagulum appears is determined. For quantitative determination, the coagulation time resulting from the solution containing factor X is read off with reference to a calibration curve obtained with a normal plasma dilution series. One unit of factor X corresponds to the factor X activity of 1 ml of normal plasma.
To destroy the hepatitis viruses, calcium ions, EDTA, glycine and sucrose and, if appropriate, antithrombin III and heparin are added to the solution and the mixture is heated.
After pasteurization (10 hours, 60xc2x0 C.) of human 1.5 plasma, following addition of sucrose (60 g/100 g) and glycine (2 mol/l) and in the presence of 25 mmol/l of calcium chloride and 5 mmol/l of EDTA, the following activities, based on the plasma employed, were found: F II 90%, F VII 85%, F IX 83%, F X 80%.
The influence the various stabilizers have on obtaining the activity of the individual prothrombin factors in a plasma of placenta fraction on heating can be seen from Table I. Pasteurization with sucrose and glycine alone gives a poor F IX yield. However, this is highly disadvantageous, since the prothrombin concentrate is chiefly used for therapy of hemophilia B. The additional use of calcium ions without a chelating agent in a concentration of 6.25 mmol/l leads to an activation of F VII and F IX and also reduces the yields of F II and F X. Such a product would be unsuitable for use on humans because of a possible risk of thrombosis. If EDTA is used without calcium ions in a concentration of 5 mmol/l, in addition to sucrose and glycine, poor F IX and F X yields are achieved, with good F II and F VII yields. The prothrombin factors cannot be stabilized merely with antithrombin III, heparin, sucrose and glycine.
The best result is given by the combination of calcium ions with EDTA with addition of antithrombin III and heparin with sucrose and glycine. Under these conditions, the yields for all four factors are of the order of about 80% and the product is also free from activated factors and thrombin.
The concentrations of coagulation factors in an aqueous solution with which the process described can be carried out are, in particular, in the following ranges: factor II: 0.3 to 50 units/ml; factor VII: 0.3 to 30 units/m; factor IX: 0.5 to 60 units/ml; and factor X: 0.4 to 60 units/m.
Particularly preferred ranges are 0.5 to 25 for factor II, 0.5 to 15 for factor VII, 0.7 to 30 for factor IX and 0.6 to 30 for factor X.
A solution which contains the active compoundsxe2x80x94i.e. in the present case the coagulation factorsxe2x80x94in concentrations which are higher than those of the natural starting materials is usually designated a concentrate.
For further purification, the heated solution can be centrifuged, if appropriate. Impurities can be removed by precipitation with ammonium sulfate and 30-45% saturation.
It is then possible to adsorb the supernatant liquor onto 0.04 to 1.0 g of calcium phosphate per 100 ml of solution, to wash the loaded adsorbent and elute it with citrate buffer and to dialyze the eluate.
For use on humans, the product can be subjected to sterilization by filtration.
The invention particularly relates to a product of factors II, VII, IX and X which is free from active viruses and can be obtained by this process.
It is advantageous to add protein-stabilizing substances, for example proteins, aminoacids or carbohydrates, to the product in order to increase the storage stability. Finally, the product subjected to this treatment can be supplied in freeze-dried form, in which case the addition of anticoagulants, such as, for example, heparin, may be advantageous.
The product according to the invention, in particular the product in the form of a concentrate, is, in a solution suitable for pharmaceutical administration, a medicament for the treatment of coagulopathy and can be used intravenously, advantageously as an infusion, for the therapy and prophylaxis of hemorrhages caused by factor deficiency.
In the form of a lyophilized, coagulation-promoting plasma, the pasteurized product covers the indications of fresh-frozen plasma, for example topping up of the blood volume, maintenance of the oncotic pressure, blood loss after major operations and accidents and treatment of various forms of shock and first aid for polytraumatized patients.